Skeletal muscle is a prime target organ for gene therapy, as engineered myoblasts can be introduced to fuse with mature muscle, forming a stable hybrid organ within the adults. The ability of injected satellite cells to fuse with myofibers directly affects the efficacy of cell-based gene therapy. Therefore, understanding the mechanisms controlling myoblast fusion promises to offer novel therapeutic possibilities in the future. We are using the fruit fly Drosophila as a genetic model to study the molecular mechanisms of myoblast fusion. Studies in recent years have revealed that many of the cellular and molecular events involved in muscle development are evolutionarily conserved between fly and vertebrates. This conservation has made it possible to dissect muscle differentiation using Drosophila genetics and to uncover essential genes required for myoblast fusion in flies and vertebrates. In a genetic screen for mutants in muscle development, we have identified a number of new loci required for myoblast fusion. To date, we have molecularly characterized two genes, antisocial and loner. Studies of these and other fusion genes have begun to reveal a signaling cascade controlling myoblast fusion. Antisocial is a novel adaptor protein that is localized to subcellular sites of fusion and relays the fusion signal from cell membrane to actin cytoskeleton. Loner, a guanine nucleotide exchange factor for ARF6, can also be recruited to sites of fusion. The goal of this project is to address two major questions concerning the mechanisms of myoblast fusion. First, how are fusion proteins recruited to sites of fusion by transmembrane receptors? Second, how is actin cytoskeletal rearrangement achieved during myoblast fusion? We propose a series of experiments to investigate the mechanisms by which Ants and Loner are targeted to sites of fusion. In Specific Aim I, we will identify domain(s) in Ants that are responsible for its localization to sites of fusion. In Specific Aim II, we will use biochemical and yeast two- hybrid approaches to isolate the intermediary protein(s) responsible for recruiting Loner to sites of fusion. In Specific Aim III, we propose the identification and characterization of a new component of the myoblast fusion machinery. We will examine its phenotype, localization, and interactions with known fusion proteins as well as with components of the actin cytoskeleton.